N,N'-(2,5-dichloro-1,4-phenylene)bis[4-[[2-chloro-5-(trifluoromethyl)phenyl]azo]-3-hydroxynaphthalene-2-carboxamide]

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-16 to 2011-12-22

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 mg/l was freshly prepared with dilution water and stirred for 48 h (1100rpm). Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). Centrifugation for 20 min at 10000 rpm was found to be insufficient.
- Eluate: Dilution water
- Differential loading: None, limit test item concentration
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

22.5 - 24.0 °C, mean value: 23.3 °C

pH:

Nominal concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.89 7.94
Control 7.90 7.97

Dissolved oxygen:

Not measured

Nominal and measured concentrations:

Nominal test concentrations: control and 1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through: None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4910 cells/mL
- Control end cells density: Mean 634285 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 69.2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.

TEST CONCENTRATIONS
- Range finding study: No

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No acute toxic effects within the range of solubility.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No acute toxic effects within the range of solubility.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations (mg/L), (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.488 0.699 ± 0.471
95 % confidence interval 0.466 – 0.512
Yield inhibition
EyC50 0.291 0.318 ± 0.147
95 % confidence interval 0.274 – 0.328

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

NOEC, LOEC, EC - values of PV-Echtbraun RL (0-72 hours)

                   based on nominal concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	> 1.00
EyC20	> 1.00
EyC50	> 1.00

Cell Densities

	Nominal concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
1.00		1		4910		29017	165338	711305
		2		4910		21642	135977	771998
		3		4910		22620	135753	676405
		4		4910		26607	173530	760929
		5		4910		27315	163950	683717
		6		4910		24681	134846	697494
		Mean		4910		25314	151566	716975
Control		1		4910		24999	142808	651898
		2		4910		23784	149659	608401
		3		4910		22508	146782	693872
		4		4910		22454	139897	566799
		5		4910		25470	140194	624886
		6		4910		26851	163111	659853
		Mean		4910		24344	147075	634285

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		1.66	-2.40		706395	-12.2
	2		1.69	-4.09		767088	-21.9
	3		1.64	-1.37		671495	-6.69
	4		1.68	-3.79		756019	-20.1
	5		1.65	-1.59		678807	-7.85
	6		1.65	-2.00		692584	-10.0
	Mean	(+)*	1.66	-2.54	(+)*	712065	-13.1
Control	1		1.63			646988
	2		1.61			603491
	3		1.65			688962
	4		1.58			561889
	5		1.62			619976
	6		1.63			654943
	Mean		1.62			629375

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.63	1.74	1.52	1.63	0.112	6.88	11.0
	2	1.58	1.84	1.40	1.61	0.220	13.7
	3	1.52	1.88	1.55	1.65	0.195	11.8
	4	1.52	1.83	1.40	1.58	0.222	14.0
	5	1.65	1.71	1.50	1.62	0.109	6.74
	6	1.70	1.80	1.40	1.63	0.211	12.9
				Mean	1.62
				SD ±	0.02
				CV [%]	1.45

Validity criteria fulfilled:

yes

Conclusions:

No acute toxic effects occured within the range of solubility. The test substance is with high probability acutely not harmful to aquatic algae.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2011-12-16 to 2011-12-22

Reliability:

1 (reliable without restriction)

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Red 242 (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 mg/l was freshly prepared with dilution water and stirred for 48 h (1100rpm). Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). Centrifugation for 20 min at 10000 rpm was found to be insufficient.
- Eluate: Dilution water
- Differential loading: None, limit test item concentration
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

22.5 - 24.0 °C, mean value: 23.3 °C

pH:

Nominal concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.89 7.94
Control 7.90 7.97

Dissolved oxygen:

Not measured

Nominal and measured concentrations:

Nominal test concentrations: control and 1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through: None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 4910 cells/mL
- Control end cells density: Mean 634285 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 69.2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.

TEST CONCENTRATIONS
- Range finding study: No

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No acute toxic effects within the range of solubility.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No acute toxic effects within the range of solubility.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations (mg/L), (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.488 0.699 ± 0.471
95 % confidence interval 0.466 – 0.512
Yield inhibition
EyC50 0.291 0.318 ± 0.147
95 % confidence interval 0.274 – 0.328

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

NOEC, LOEC, EC - values of PV-Echtbraun RL (0-72 hours)

                   based on nominal concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield
NOEC	1.00
LOEC	> 1.00
EyC10	> 1.00
EyC20	> 1.00
EyC50	> 1.00

Cell Densities

	Nominal concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
1.00		1		4910		29017	165338	711305
		2		4910		21642	135977	771998
		3		4910		22620	135753	676405
		4		4910		26607	173530	760929
		5		4910		27315	163950	683717
		6		4910		24681	134846	697494
		Mean		4910		25314	151566	716975
Control		1		4910		24999	142808	651898
		2		4910		23784	149659	608401
		3		4910		22508	146782	693872
		4		4910		22454	139897	566799
		5		4910		25470	140194	624886
		6		4910		26851	163111	659853
		Mean		4910		24344	147075	634285

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		1.66	-2.40		706395	-12.2
	2		1.69	-4.09		767088	-21.9
	3		1.64	-1.37		671495	-6.69
	4		1.68	-3.79		756019	-20.1
	5		1.65	-1.59		678807	-7.85
	6		1.65	-2.00		692584	-10.0
	Mean	(+)*	1.66	-2.54	(+)*	712065	-13.1
Control	1		1.63			646988
	2		1.61			603491
	3		1.65			688962
	4		1.58			561889
	5		1.62			619976
	6		1.63			654943
	Mean		1.62			629375

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.63	1.74	1.52	1.63	0.112	6.88	11.0
	2	1.58	1.84	1.40	1.61	0.220	13.7
	3	1.52	1.88	1.55	1.65	0.195	11.8
	4	1.52	1.83	1.40	1.58	0.222	14.0
	5	1.65	1.71	1.50	1.62	0.109	6.74
	6	1.70	1.80	1.40	1.63	0.211	12.9
				Mean	1.62
				SD ±	0.02
				CV [%]	1.45

Validity criteria fulfilled:

yes

Conclusions:

No acute toxic effects occured within the range of solubility. The test substance is with high probability acutely not harmful to aquatic algae.

Description of key information

Red disazo condensation pigments are with high probability not harmful to aquatic algae. 
No toxic effects occur at the range of the substances solubility.
EC50 (72h) and EC10 (72h) >= 1 mg/L (nominal) for growth rate (OECD 201)

Additional information

The toxicity on aquatic algae was investigated for two ‘red disazo condensation pigments’(CAS 68516-75-6 and CAS 71566-54-6) [DR.U.NOACK-LABORATORIEN, 2012; lnstitute of lndustrial Organic Chemistry, Branch Pszczyna, 2012]. The studies were conducted with the green algae species Desmodesmus subspicatus (CAS 68516-75-6) and Pseudokirchneriella subcapitata (CAS 71566-54-6). Based on the low water solubility of the test item, saturated stock solutions were prepared and the tests were conducted as limit tests with nominal concentrations of 1 mg/L (CAS 68516-75-6) and 10 mg/L (CAS 71566-54-6), respectively. After 72 hours of exposure no toxic effects on algae observed, neither in the study with Desmodesmus subspicatus nor in the study with Pseudokirchneriella subcapitata.